Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO₂, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (F_AF_B), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s⁻¹). These data unambiguously show that reduced ferredoxin, or possibly reduced F_AF_B, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (F_AF_B) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.     

Photosystem I with benzoquinone analogues incorporated into the A<Subscript>1</Subscript> binding site

Time-resolved FTIR difference spectroscopy has been used to study photosystem I (PSI) particles with three different benzoquinones [plastoquinone-9 (PQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (Cl₄BQ)] incorporated into the A₁ binding site. If PSI samples are cooled in the dark to 77 K, the incorporated benzoquinones are shown to be functional, allowing the production of time-resolved (P700⁺A₁^??P700A₁) FTIR difference spectra. If samples are subjected to repetitive flash illumination at room temperature prior to cooling, however, the time-resolved FTIR difference spectra at 77 K display contributions typical of the P700 triplet state (³P700), indicating a loss of functionality of the incorporated benzoquinones, that occurs because of double protonation of the incorporated benzoquinones. The benzoquinone protonation mechanism likely involves nearby water molecules but does not involve the terminal iron–sulfur clusters F_A and F_B. These results and conclusions resolve discrepancies between results from previous low-temperature FTIR and EPR studies on similar PSI samples with PQ incorporated.     

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, F_A and F_B. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700⁺ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to F_A and F_B were absent in the mutant strain, but a reversible F_x signal was present with broad resonances at g=2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g=2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of F_A ^- and F_B ^-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700⁺ [F_A/F_B]^- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to F_x.     

Heat-Stability of Iron-Sulfur Centers and P-700 in Photosystem I Reaction Center Complexes Isolated from the Thermophilic Cyanobacterium Synechococcus elongatus

Stabilities of iron-sulfur centers and reaction center chlorophyllP-700 in Photosystem I reaction center complex (CP1-a), isolatedby sodium dodecyl sulfate treatment from the thermophilic cyanobacteriumSynechococcus elongatus, were studied by EPR and optical spectroscopy.P-700 was destroyed by treatment at temperatures above 80?Cfor 5 minutes with a half inactivation temperature of 93?C.The three iron-sulfur centers F_A, F_B and F_X showed similar thermalstabilities and were half inactivated at about 70?C. Thus, theisolated Photosystem I reaction center complexes of S. elongatusare still highly resistant to heat. (Received May 9, 1990; Accepted June 25, 1990)     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Protection against photoinhibition in the alpine plant Geum montanum

Geum montanum L. is an alpine plant usually found at altitudes between 1700 and 2600 m. Its wintergreen leaves can be subjected to very low temperatures and at the same time receive high photon flux densities at the beginning of the growth season when the snow melts. We report results of a study, performed with classical methods of biophysics, showing that leaves of G. montanum were remarkably tolerant to sunlight even at low temperatures. This tolerance results from the interplay of photorespiration and CO₂ photosassimilation. When temperatures approach 0°C, responses include stomatal opening and CO₂ uptake even under desiccation stress. This permits linear electron transport that is sufficient to avoid the excessive reduction of the electron transport chain which is known to lead to photodamage. In addition, excitation energy was shifted from photosystem (PS)II to PSI which is a very efficient energy quencher. Sensitivity of P700 in PSI to oxidation by far-red light was decreased and rates of dark reduction of photooxidized P700 were increased by actinic illumination, suggesting activation of cyclic electron transport. Consistent with this, far-red light was able to decrease the quantum yield of PSII (measured by the F _v/F _m ratio of chlorophyll fluorescence). We suggest that cyclic electron transport decreases the lumenal pH under strong light. In the presence of zeaxanthin, this increases energy dissipation at the PSII level. At low temperatures, P700 remained strongly oxidized under high irradiation while the primary electron acceptor of PSII, Q_A, was largely reduced. This shows efficient control of electron transport presumably at the level of the cytochrome b/f complex and suggests formation of a protective transthylakoid proton gradient even when linear electron transport is much reduced in the cold. Thus, several mechanisms cooperate to effectively protect the photosynthetic apparatus of G. montanum from photodamage. We see no indication of destructive “photostress” in this species during the growth season under alpine low-temperature and drought conditions. Received: 2 March 1998 / Accepted: 7 January 1999     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Reduction of the primary donor P700 of photosystem I during steady-state photosynthesis under low light in <Emphasis Type="Italic">Arabidopsis</Emphasis>

During steady-state photosynthesis in low-light, 830-nm absorption (A₈₃₀) by leaves was close to that in darkness in Arabidopsis, indicating that the primary donor P700 in the reaction center of photosystem I (PSI) was in reduced form. However, P700 was not fully oxidized by a saturating light pulse, suggesting the presence of a population of PSI centers with reduced P700 that remains thermodynamically stable during the application of the saturating light pulse (i.e., reduced-inactive P700). To substantiate this, the effects of methyl viologen (MV) and far-red light on P700 oxidation by the saturating light pulse were analyzed, and the cumulative effects of repetitive application of the saturating light pulse on photosynthesis were analyzed using a mutant crr2-2 with impaired PSI cyclic electron flow. We concluded that the reduced-inactive P700 in low-light as revealed by saturating light pulse indicates limitations of electron flow at the PSI acceptor side.     

Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I

To study electrogenesis the photosystem I particles fromSynechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes adsorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center F_x reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers F_x and F_A/F_B, and (iii) reduction of photo-oxidized P700⁺ by reduced cytochromec ₅₅₃. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).     

Multiple pathways of charge recombination revealed by the temperature dependence of electron transfer kinetics in cyanobacterial photosystem I

The kinetics of charge recombination in Photosystem I P₇₀₀-F_A/F_B complexes and P₇₀₀-F_X cores lacking the terminal iron?sulfur clusters were studied over a temperatures range of 310 K to 4.2 K. Analysis of the charge recombination kinetics in this temperature range allowed the assignment of backward electron transfer from the different electron acceptors to P₇₀₀⁺. The kinetic and thermodynamic parameters of these recombination reactions were determined. The kinetics of all electron transfer reactions were activation-less below 170 K, the glass transition temperature of the water-glycerol solution. Above this temperature, recombination from [F_A/F_B]^? in P₇₀₀-F_A/F_B complexes was found to proceed along two pathways with different activation energies (E_a). The charge recombination via A_1A has an E_a of ~290 meV and is dominant at temperatures above ~280 K, whereas the direct recombination from F_X^? has an E_a of 22 meV and is prevalent in the 200 K to 270 K temperature range. Charge recombination from the F_X cluster becomes highly heterogeneous at temperatures below 200 K. The conformational mobility of Photosystem I was studied by molecular dynamics simulations. The F_X cluster was found to ‘swing’ by ~30° along the axis between the two sulfur atoms proximal to F_A/F_B. The partial rotation of F_X is accompanied by significant changes of electric potential within the iron?sulfur cluster, which may induce preferential electron localization at different atoms of the F_X cluster. These effects may account for the partial arrest of forward electron transfer and for the heterogeneity of charge recombination observed at the glass transition temperature.     

Critical evaluation of electron transfer kinetics in P700–FA/FB, P700–FX, and P700–A1 Photosystem I core complexes in liquid and in trehalose glass

This work aims to fully elucidate the effects of a trehalose glassy matrix on electron transfer reactions in cyanobacterial Photosystem I (PS I). Forward and backward electron transfer rates from A_1A^? and A_1B^? to F_X, and charge recombination rates from A₀^?, A_1B^?, A_1A^?, F_X^?, and [F_A/F_B]^? to P₇₀₀⁺ were measured in P₇₀₀–F_A/F_B complexes, P₇₀₀–F_X cores, and P₇₀₀–A₁ cores, both in liquid and in a trehalose glassy matrix at 11% humidity. By comparing CONTIN-resolved kinetic events over 6 orders of time in increasingly simplified versions of PS I at 480?nm, a wavelength that reports primarily A_1A^?/A_1B^? oxidation, and over 9 orders of time at 830?nm, a wavelength that reports P₇₀₀⁺ reduction and A₀^? oxidation, assignments could be made for nearly all of the resolved kinetic phases. Trehalose-embedded PS I samples demonstrated partially arrested forward electron transfer. The fractions of complexes in which electron transfer did not proceed beyond A₀, A₁ and F_X were 53%, 16% and 22%, respectively, with only 10% of electrons reaching the terminal F_A/F_B clusters. The ~10?μs and ~150?μs components in both liquid and trehalose-embedded PS I were assigned to recombination between A_1B^? and P₇₀₀⁺ and between A_1A^? and P₇₀₀⁺, respectively. The kinetics and amplitudes of these resolved kinetic phases in liquid and trehalose-embedded PS I samples could be well-fitted by a kinetic model that allowed us to calculate the asymmetrical contribution of the A_1A^? and A_1B^? quinones to the electrochromic signal at 480?nm. Possible reasons for these effects are discussed.     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

Kinetic modeling of electron transfer reactions in photosystem I complexes of various structures with substituted quinone acceptors

The reduction kinetics of the photo-oxidized primary electron donor P₇₀₀ in photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 were analyzed within the kinetic model, which considers electron transfer (ET) reactions between P₇₀₀, secondary quinone acceptor A₁, iron-sulfur clusters and external electron donor and acceptors – methylviologen (MV), 2,3-dichloro-naphthoquinone (Cl₂NQ) and oxygen. PS I complexes containing various quinones in the A₁-binding site (phylloquinone PhQ, plastoquinone-9 PQ and Cl₂NQ) as well as F _X-core complexes, depleted of terminal iron–sulfur F _A/F _B clusters, were studied. The acceleration of charge recombination in F _X-core complexes by PhQ/PQ substitution indicates that backward ET from the iron–sulfur clusters involves quinone in the A₁-binding site. The kinetic parameters of ET reactions were obtained by global fitting of the P₇₀₀ ⁺ reduction with the kinetic model. The free energy gap ΔG ₀ between F _X and F _A/F _B clusters was estimated as ?130 meV. The driving force of ET from A₁ to F _X was determined as ?50 and ?220 meV for PhQ in the A and B cofactor branches, respectively. For PQ in A_1A-site, this reaction was found to be endergonic (ΔG ₀?=?+75 meV). The interaction of PS I with external acceptors was quantitatively described in terms of Michaelis–Menten kinetics. The second-order rate constants of ET from F _A/F _B, F _X and Cl₂NQ in the A₁-site of PS I to external acceptors were estimated. The side production of superoxide radical in the A₁-site by oxygen reduction via the Mehler reaction might comprise ≥0.3% of the total electron flow in PS I.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Purification and crystallization of Photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002

An active photosystem (PSI) complex was isolated from a phycobilisome-less mutant of the mesophilic cyanobacterium Synechococcus PCC 7002 by a mild procedure. Purification of PS I was achieved using a sucrose density gradient and an isoelectric focussing subsequent to the extraction of PSI from thylakoids with dodecyl--maltoside. Electron microscopy and gel filtration HPLC suggested that the isolated complex represents a trimeric form of PSI. The trimeric form was resistant to pH or detergent exchange. A molecular weight of 690 kDa to 760 kDa has been determined for the complex by gel filtration HPLC in several detergents or mixtures of detergents.The PSI complex contains the polypeptides of the psaA, psaB, psaC, psaD, psaE, psaL gene products and two small polypeptides as determined by SDS-PAGE and N-terminal sequencing; its antenna size is 77±2 Chl a/P₇₀₀. The full set of Fe-S clusters (F_A, F_B and F_X) was observed by EPR-spectroscopy. A preliminary characterization of crystals obtained from this preparation was carried out using SDS-PAGE, optical and EPR spectroscopy.Abbreviations BA benzamidine - CAS 6-amino-n-caproic acid - C₈-G octyl--D-glucopyranoside - C₁₂-M lauryl--D-maltoside - C₁₀-M decyl--D-maltoside - C₈-TG octyl--D-thioglucoside - Chl a chlorophyll a - EPR electron paramagnetic resonance - F_A, F_B, F_X iron-sulfur centers - HPLC high perfomance liquid chromatography - kDa kilodalton(s) - LDAO lauryldimethylamine oxide - MES 2-(N-morpholino)ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - P₇₀₀ primary electron donor - SB12 sulfobetain 12 - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tris tris(hydroxymethyl)-aminomethane     

Recovery of photosystem I and II activities during re-hydration of lichen<Emphasis Type="Italic"> Hypogymnia physodes</Emphasis> thalli

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700⁺ and the reduced acceptor F_X of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700⁺ reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q_A^–, the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.Abbreviations A₀ and A₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - Chl a Chlorophyll a - F_m Maximal level of chlorophyll fluorescence when all PSII centers are closed - F_o Minimal level of fluorescence when all PSII centers are open after dark adaptation - FR Far-red - F_v Variable fluorescence (=F_m–F_o) - F_X, F_A, and F_B Iron–sulfur centers - MT pulse Multiple-turnover pulse - PS Photosystem - P700 Reaction center chlorophyll of PSI - Q_A Primary quinone acceptor of PSII - Q_B Secondary quinone acceptor of PSII - ST pulse Single-turnover pulse     

Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c₆ (cyt. c₆), and Fd. It is shown that cyt. c₆ oxidation contributes to the NIR absorption changes. The reduction of (F_AF_B), the terminal electron acceptor of PSI, was also observed and the shape of the (F_AF_B) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c₆, reduced minus oxidized for (F_AF_B), and Fd) were determined with values of 0.207?±?0.004, –?0.033?±?0.006, –?0.036?±?0.008, and –?0.021?±?0.005 for PCy, cyt. c₆, (F_AF_B) (single reduction), and Fd, respectively, by taking a reference value of +?1 for P700⁺. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.